Reference control data obtained from an in vivo comet-micronucleus combination assay using Sprague Dawley rats

According to the International Conference on Harmonization Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (ICH S2(R1)), a positive response in any in vitro assay necessitates additional in vivo test(s) (other tissue/endpoint) in addition to the erythrocyte micronucleus test when Option 1 of the test battery is selected. When Option 2 of the test battery is selected, a bacterial gene mutation test and two in vivo tests with different tissues/endpoint are required. The in vivo alkaline comet assay is recommended as the second in vivo test because it can detect a broad spectrum of DNA damage in any tissue and can be combined with the erythrocyte micronucleus test. Considering animal welfare, a combination assay is preferable to an individual assay. Thus, we validated the protocol for the in vivo comet-micronucleus combination assay in rats with three daily administrations and determined the dose of the positive control (ethyl methanesulfonate; EMS, 200 mg/kg/day). We also collected the negative control (vehicle) and positive control (EMS) data from the comet (liver, stomach, and kidney) and micronucleus (bone marrow) combination assay using male Sprague Dawley (SD) rats. The negative control data were comparable to our historical control data obtained from stand-alone assays. The positive control data showed clear and consistent positive responses in both endpoints.     

Caryocar brasiliense camb protects against genomic and oxidative damage in urethane-induced lung carcinogenesis

The antioxidant effects of Caryocar brasiliense Camb, commonly known as the pequi fruit, have not been evaluated to determine their protective effects against oxidative damage in lung carcinogenesis. In the present study, we evaluated the role of pequi fruit against urethane-induced DNA damage and oxidative stress in forty 8-12 week old male BALB/C mice. An in vivo comet assay was performed to assess DNA damage in lung tissues and changes in lipid peroxidation and redox cycle antioxidants were monitored for oxidative stress. Prior supplementation with pequi oil or its extract (15 µL, 60 days) significantly reduced urethane-induced oxidative stress. A protective effect against DNA damage was associated with the modulation of lipid peroxidation and low protein and gene expression of nitric oxide synthase. These findings suggest that the intake of pequi fruit might protect against in vivo genotoxicity and oxidative stress.     

Cardioprotection against experimental myocardial ischemic injury using cornin

Phosphorylated-cyclic adenosine monophosphate response element-binding protein (Phospho-CREB) has an important role in the pathogenesis of myocardial ischemia. We isolated the iridoid glycoside cornin from the fruit of Verbena officinalis L, investigated its effects against myocardial ischemia and reperfusion (I/R) injury in vivo, and elucidated its potential mechanism in vitro. Effects of cornin on cell viability, as well as expression of phospho-CREB and phospho-Akt in hypoxic H9c2 cells in vitro, and myocardial I/R injury in vivo, were investigated. Cornin attenuated hypoxia-induced cytotoxicity significantly in H9c2 cells in a concentration-dependent manner. Treatment of H9c2 cells with cornin (10 µM) blocked the reduction of expression of phospho-CREB and phospho-Akt in a hypoxic condition. Treatment of rats with cornin (30 mg/kg, iv) protected them from myocardial I/R injury as indicated by a decrease in infarct volume, improvement in hemodynamics, and reduction of severity of myocardial damage. Cornin treatment also attenuated the reduction of expression of phospho-CREB and phospho-Akt in ischemic myocardial tissue. These data suggest that cornin exerts protective effects due to an increase in expression of phospho-CREB and phospho-Akt.     

Cytogenetic studies of PCB77 on brown trout (Salmo trutta fario) using the micronucleus test and the alkaline comet assay

Polychlorinated biphenyls (PCBs) are stable pollutants, whichcan be found in almost every compartment of terrestrial andaquatic ecosystems. They are very lipophilic and therefore havethe potency of accumulating in the fat stores of animals. Themechanisms by which PCBs exert their adverse effects are stillunclear. It is known that PCBs induce some important biotransformationenzymes, but their mutagenic properties are still controversial.The DNA breakage and clastogenic potency of a planar PCB77 (3,3', 4, 4'-tetrachlorobiphenyl) was determined in vivo in fish,using the single cell gel electrophoresis or comet assay andthe micronucleus test, on erythrocytes of the brown trout exposedfor 3, 9 and 14 days to initial PCB concentrations of 780 and918 pg/ml, dissolved in the water. Blood was taken by a caudalpuncture and the erythrocytes were either deposited in an agarosegel (0.6%) for the comet assay or smeared directly on slidesfor the micronucleus test. Five fish were studied per treatmentand 50 and 2000 erythrocytes per concentration and per animalwere analysed for the comet assay and the micronucleus testrespectively. Ethyl methanesulphonate (EMS) at a concentrationof 25 mg/I water was used as a positive control. Although EMSinduced a statistically significant increase of single strandbreaks in the comet assay, in neither of the two tests used,were mutagenic effects due to PCB exposure observed. ³To whom correspondence should be addressed     

FTY720 induces apoptosis in B16F10-NEX2 murine melanoma cells,limits metastatic development in vivo,and modulates the immune system

OBJECTIVE:

Available chemotherapy presents poor control over the development of metastatic melanoma. FTY720 is a compound already approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis. It has also been observed that FTY720 inhibits tumor growth in vivo (experimental models) and in vitro (animal and human tumor cells). The aim of this study was to evaluate the effects of FTY720 on a metastatic melanoma model and in tumor cell lines.

METHODS:

We analyzed FTY720 efficacy in vivo in a syngeneic murine metastatic melanoma model, in which we injected tumor cells intravenously into C57BL/6 mice and then treated the mice orally with the compound for 7 days. We also treated mice and human tumor cell lines with FTY720 in vitro, and cell viability and death pathways were analyzed.

RESULTS:

FTY720 treatment limited metastatic melanoma growth in vivo and promoted a dose-dependent decrease in the viability of murine and human tumor cells in vitro. Melanoma cells treated with FTY720 exhibited characteristics of programmed cell death, reactive oxygen species generation, and increased β-catenin expression. In addition, FTY720 treatment resulted in an immunomodulatory effect in vivo by decreasing the percentage of Foxp3+ cells, without interfering with CD8+ T cells or lymphocyte-producing interferon-gamma.

CONCLUSION:

Further studies are needed using FTY720 as a monotherapy or in combined therapy, as different types of cancer cells would require a variety of signaling pathways to be extinguished.     

Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract.     

Superiority of corpuscular BCG to soluble PPD antigen in the leucocyte migration assay

The recognized trend towards the standardization of the leucocyte migration assay prompted the comparison of efficacy of BCG with soluble purified protein derivative (PPD) antigens in this test. Even low BCG concentrations permit high correlations between in vivo and in vitro responses, whilst PPD doses needed to elicit comparable reactions fall within toxic concentrations. BCG-induced inhibition of leucocyte migration is abolished by the protein synthesis inhibitor, cycloheximide.     

RNAi-mediated knockdown of FANCF suppresses cell proliferation,migration, invasion,and drug resistance potential of breast cancer cells

Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.     

Nanoparticle toxicity assessment using an in vitro 3-D kidney organoid culture model

Nanocarriers and nanoparticles remain an intense pharmaceutical and medical imaging technology interest. Their entry into clinical use is hampered by the lack of reliable in vitro models that accurately predict in vivo toxicity. This study evaluates a 3-D kidney organoid proximal tubule culture to assess in vitro toxicity of the hydroxylated generation-5 PAMAM dendrimer (G5-OH) compared to previously published preclinical in vivo rodent nephrotoxicity data. 3-D kidney proximal tubule cultures were created using isolated murine proximal tubule fractions suspended in a biomedical grade hyaluronic acid-based hydrogel. Toxicity in these cultures to neutral G5-OH dendrimer nanoparticles and gold nanoparticles in vitro was assessed using clinical biomarker generation. Neutral PAMAM nanoparticle dendrimers elicit in vivo-relevant kidney biomarkers and cell viability in a 3-D kidney organoid culture that closely reflect toxicity markers reported in vivo in rodent nephrotoxicity models exposed to this same nanoparticle.     

Bone marrow genotoxicity of 2,5‐dimethylfuran,a green biofuel candidate

2,5‐Dimethylfuran (DMF) is being considered as a potential green transportation biofuel, but there is limited information about its toxicity and safety. We examined DMF toxicity in the bone marrow using a murine in vitro erythropoietic micronucleus assay and found that exposure to DMF (0.1 mM, 1 hr) induced an increase in micronuclei frequency compared with controls. These data suggest that DMF may be genotoxic to hematopoietic cells and that more thorough toxicological studies on DMF should be conducted to ensure public and occupational safety before it is considered a viable biofuel and produced in mass quantities. As well as specific data on DMF, our study further validates an in vitro cell culture system that captures the essential features of the in vivo mammalian micronucleus genotoxicity assay, enabling increased throughput and controlled studies on hematopoietic DNA damage response, while reducing animal sacrifice. In vitro assays, such as the in vitro micronucleus assay, will be essential as international chemical policy is increasingly utilizing green chemistry principles that require more toxicological testing. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Ex vivo non-invasive assessment of cell viability and proliferation in bio-engineered whole organ constructs

Decellularized organ scaffolds allow whole organ regeneration and study of cell behavior in three-dimensional culture conditions. Cell viability within the bio-engineered organ constructs is an essential parameter reflecting the performance of participating cells during long-term ex vivo culture, and is a prerequisite for further functional performance. Resazurin-based redox metabolic assays have been used to monitor cell viability in both two- and three-dimensional cell cultures. Here we developed a method for monitoring cell viability and proliferation in bio-engineered organ constructs using a resazurin perfusion assay. This method allows non-invasive, repetitive and rapid estimation of viable cell numbers during long-term ex vivo culture. As a proof-of-principle, we assessed the performance of two different endothelial sources and the impact of different perfusion programs on endothelial viability after re-endothelialization of decellularized lung scaffolds. The resazurin-based perfusion assay revealed changes in endothelial viability and proliferation during long-term ex vivo culture, which was consistent with histological assessment at different time points. Finally, we showed that this method could be used for assessment of proliferation and cytotoxicity after pharmacological treatment on a three-dimensional non-small cell lung cancer culture model.     

Assessment of DNA damage in lymphocytes of workers exposed to X-radiation using the micronucleus test and the comet assay.

The mutagenic and carcinogenic effects of genotoxic agents on exposed people have constituted an increasing concern. Therefore, the objective of this work was to assess DNA damage in lymphocytes of workers exposed to X-radiation using the cytokinesis-blocked micronucleus test and the comet assay (single-cell gel electrophoresis), and to compare these two techniques in the monitoring of exposed populations. The cytokinesis-blocked micronucleus test and the comet assay were employed in the monitoring of 22 workers occupationally exposed to X-radiation in a hospital in southern Brazil. The frequency of dicentric bridges was also measured. The results of both assays and the frequency of dicentric bridges revealed a significant increase in genetic effects on the cells of exposed individuals. Age was significantly correlated with micronucleus frequency and damage index in the comet assay. The concomitant analysis of dicentric bridges when determining micronucleus frequency does not require much extra work, and may serve as a reference to the type of mutagenic effect (clastogenic or aneugenic). The combination of the alkaline comet assay with the cytokinesis-blocked micronucleus test appears to be very informative for the monitoring of populations chronically exposed to genotoxic agents.     

Development of an <Emphasis Type="Italic">In Vitro</Emphasis> 3D Brain Tissue Model Mimicking <Emphasis Type="Italic">In Vivo</Emphasis>-Like Pro-inflammatory and Pro-oxidative Responses

To analyze complex inflammatory responses in an in vitro system, we constructed a new 3D in vitro brain tissue model that exhibits in vivo-like tissue responses (e.g. immune cell phenotypes, and molecular response) to inflammatory stimuli. Finite element modeling of oxygen diffusion and cellular oxygen consumption predicted the oxygen profile within 3D structures, consisting of Type I collagen hydrogel embedded with murine microglia. Viability and cytotoxicity analyses supported the mathematical analysis, determining optimal cell growth conditions for 3D construct development. Real-time RT-PCR and ELISA demonstrated significant up-regulation of pro-inflammatory mediators, such as TNF-α, MCP-1, IL-6 and IL-1β, in lipopolysaccharide (LPS)-stimulated in vitro cell culture (2D and 3D) and in vivo mouse model systems. Interestingly, levels of inflammatory responses from the in vitro 3D model system were more similar to in vivo than in vitro 2D. Additionally, in situ dihydroethidium (DHE) assay and immunofluorescence staining revealed that levels of LPS-stimulated reactive oxygen species (ROS) generation and microglial activation from in vitro 3D model system were closer to in vivo than in vitro 2D. These results demonstrated that an in vitro 3D model provides more physiologically relevant pro-oxidative and pro-inflammatory environments in brain than an in vitro 2D model.     

Can the comet assay be used reliably to detect nanoparticle‐induced genotoxicity?

The comet assay is a sensitive method to detect DNA strand breaks as well as oxidatively damaged DNA at the level of single cells. Today the assay is commonly used in nano‐genotoxicology. In this review we critically discuss possible interactions between nanoparticles (NPs) and the comet assay. Concerns for such interactions have arisen from the occasional observation of NPs in the “comet head”, which implies that NPs may be present while the assay is being performed. This could give rise to false positive or false negative results, depending on the type of comet assay endpoint and NP. For most NPs, an interaction that substantially impacts the comet assay results is unlikely. For photocatalytically active NPs such as TiO₂, on the other hand, exposure to light containing UV can lead to increased DNA damage. Samples should therefore not be exposed to such light. By comparing studies in which both the comet assay and the micronucleus assay have been used, a good consistency between the assays was found in general (69%); consistency was even higher when excluding studies on TiO₂ NPs (81%). The strong consistency between the comet and micronucleus assays for a range of different NPs—even though the two tests measure different endpoints—implies that both can be trusted in assessing the genotoxicity of NPs, and that both could be useful in a standard battery of test methods. Environ. Mol. Mutagen. 56:82–96, 2015. © 2014 Wiley Periodicals, Inc.     

Genotoxicity and cytotoxicity of sevoflurane in two human cell lines in vitro with ionizing radiation

Objective:

To determine the in vitro toxicity of different concentrations of sevoflurane in cells exposed to X-ray.

Methods:

The genotoxic effects of sevofluorane were studied by means of the micronucleus test in cytokinesis-blocked cells of irradiated human lymphocytes. Subsequently, its cytotoxic effects on PNT2 (normal prostate) cells was determined using the cell viability test (MTT) and compared with those induced by different doses of X-rays.

Results:

A dose- and time-dependent cytotoxic effect of sevofluorane on PNT2 cells was determined (p >0.001) and a dose-dependent genotoxic effect of sevofluorane was established (p >0.001). Hovewer, at volumes lower than 30 μL of sevofluorane at 100%, a non-toxic effect on PNT2 cells was shown.

Conclusion:

Sevofluorane demonstrates a genotoxic capacity as determined in vitro by micronucleus test in cytokinesis-blocked cells of irradiated human lymphocytes.     

Reversible mitotic and metabolic inhibition following the encapsulation of fibroblasts in alginate hydrogels

Limiting cell proliferation without reducing cell viability for in vivo tissue engineering applications is important in co-culture applications where the growth of one cell type must be inhibited to prevent overgrowth of the scaffold at the expense of another cell type. Also, it is vital for maintaining viability of cells in large constructs before vascularisation occurs. In this study we have shown by means of the Thiazolyl blue (MTT) assay and immuno-staining for proliferating cell nuclear antigen (PCNA) that encapsulating fibroblasts in 2% and 5% w/v calcium-alginate at a density of 7.5 × 10⁵ cells/ml as uniformly dispersed entities, enabled cells to maintain viability and caused a reversible mitotic inhibition. Alginate encapsulation also caused reversible metabolic inhibition as demonstrated by the MTT assay and fluorescent staining for mitochondrial membrane potential. Histological evaluation of the alginate constructs containing fibroblasts showed that mitotic and metabolic inhibition was possibly due to cell isolation during the first five weeks of culture. The alginate scaffold degraded with time releasing encapsulated fibroblasts. Upon implantation to a wound site this should ensure that encapsulated cells are able to replace the damaged tissue after sufficient proliferation of the co-cultured cell type or sufficient vascularisation of the construct.     

Analysis of micronucleus induction of pyrimethamine in in vitro CHL cells and in in vivo mouse bone marrow cells

In vivo and in vitro mutagenicity of pyrimethamine were examinedin the micronucleus test. Pyrimethamine strongly induced micronucleiin a dose-dependent manner in the in vitro micronucleus testusing the Chinese hamster lung (CHL) cell line, when treatedat 0.2–1.6 µg/ml for 48 h. The in vivo micronucleustest was carried out in mice after the first, second, thirdand fourth administration of doses up to 40 mg/kg p.o. The resultsshowed no increased frequency of micronuclei after any treatment,though pyrimethamine was shown to persist at levels >2 µ/mlin plasma after a single oral administration of 50 mg/kg. ¹To whom correspondence should be addressed     

Assessing testicular germ cell DNA damage in the comet assay; introduction of a proof-of-concept

The in vivo comet assay is widely used to measure genotoxicity; however, the current OECD test guideline (TG 489) does not recommend using the assay to assess testicular germ cells, due to the presence of testicular somatic cells. An adapted approach to specifically assess testicular germ cells within the comet assay is certainly warranted, considering regulatory needs for germ cell-specific genotoxicity data in relation to the increasing global production of and exposure to potentially hazardous chemicals. Here, we provide a proof-of-concept to selectively analyze round spermatids and primary spermatocytes, distinguishing them from other cells of the testicle. Utilizing the comet assay recordings of DNA content (total fluorescence intensity) and DNA damage (% tail intensity) of individual comets, we developed a framework to distinguish testicular cell populations based on differences in DNA content/ploidy and appearance. Haploid round spermatid comets are identified through (1) visual inspection of DNA content distributions, (2) setting DNA content thresholds, and (3) modeling DNA content distributions using a normal mixture distribution function. We also describe an approach to distinguish primary spermatocytes during comet scoring, based on their high DNA content and large physical size. Our concept allows both somatic and germ cells to be analyzed in the same animal, adding a versatile, sensitive, rapid, and resource-efficient assay to the limited genotoxicity assessment toolbox for germ cells. An adaptation of TG 489 facilitates accumulation of valuable information regarding distribution of substances to germ cells and their potential for inducing germ cell gene mutations and structural chromosomal aberrations.     

In vitro-in vivo Pharmacokinetic correlation model for quality assurance of antiretroviral drugs

Introduction:

The in vitro-in vivo pharmacokinetic correlation models (IVIVC) are a fundamental part of the drug discovery and development process. The ability to accurately predict the in vivo pharmacokinetic profile of a drug based on in vitro observations can have several applications during a successful development process.

Objective:

To develop a comprehensive model to predict the in vivo absorption of antiretroviral drugs based on permeability studies, in vitro and in vivo solubility and demonstrate its correlation with the pharmacokinetic profile in humans.

Methods:

Analytical tools to test the biopharmaceutical properties of stavudine, lamivudine y zidovudine were developed. The kinetics of dissolution, permeability in caco-2 cells and pharmacokinetics of absorption in rabbits and healthy volunteers were evaluated.

Results:

The cumulative areas under the curve (AUC) obtained in the permeability study with Caco-2 cells, the dissolution study and the pharmacokinetics in rabbits correlated with the cumulative AUC values in humans. These results demonstrated a direct relation between in vitro data and absorption, both in humans and in the in vivo model.

Conclusions:

The analytical methods and procedures applied to the development of an IVIVC model showed a strong correlation among themselves. These IVIVC models are proposed as alternative and cost/effective methods to evaluate the biopharmaceutical properties that determine the bioavailability of a drug and their application includes the development process, quality assurance, bioequivalence studies and pharmacosurveillance.